LOCUS       sequence-193572-        6073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a clathrin ""hook"" mutant in mammalian cells to hot-wire
            endocytosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6073)
  AUTHORS   Wood LA, Larocque G, Clarke NI, Sarkar S, Royle SJ
  TITLE     New tools for ""hot-wiring"" clathrin-mediated endocytosis with 
            temporal and spatial precision.
  JOURNAL   J Cell Biol. 2017 Sep 27. pii: jcb.201702188. doi: 
            10.1083/jcb.201702188.
  PUBMED    28954824
REFERENCE   2  (bases 1 to 6073)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol. 2017 Sep 27. pii: jcb.201702188. doi: 10.1083/jcb.201702188."
COMMENT     Sequence Label: FKBP-beta2(616-951)-GFP ∆CBM
FEATURES             Location/Qualifiers
     source          1..6073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        59..362
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        363..566
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     517..537
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(664..666,667..987)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      1012..1021
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2011..2020
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2017..2019,2020..2022,2023..2736)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2017..2019,2020..2022,2023..2733)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2062..2083)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2323..2342)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2670..2691
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2859..2980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2896..2915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2950..2969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2987..3442)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3124..3145)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3336..3355
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3469..3573
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3575..3932
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3575..3766
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3598..3618)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3783..3918
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3967..4761
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3967..4761
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4021..4040)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4631..4650
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4949..4968)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4993..5040
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5369..5957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5858..5877
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
       61 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      121 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      181 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      241 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      301 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      361 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      421 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      481 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      541 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
      601 ggactcagat ctcgagagat cgatgtgaat gggggccctg aacccgccaa gaacctgtac
      661 accatgggag tgcaggtgga aaccatctcc ccaggagacg ggcgcacctt ccccaagcgc
      721 ggccagacct gcgtggtgca ctacaccggg atgcttgaag atggaaagaa atttgattcc
      781 tcccgggaca gaaacaagcc ctttaagttt atgctaggca agcaggaggt gatccgaggc
      841 tgggaagaag gggttgccca gatgagtgtg ggtcagagag ccaaactgac tatatctcca
      901 gattatgcct atggtgccac tgggcaccca ggcatcatcc caccacatgc cactctcgtc
      961 ttcgatgtgg agcttctaaa actggaacgg taccgcgggc ccgggatccc cgccaccatg
     1021 gaacagcctc aggttatccc ctctcaaggt gatctcggtc ccccagtcaa tgtgccacag
     1081 gtgtcctcca tgcagatggg agcagtggat ctcctaggag gaggactaga tagtctgctt
     1141 ggcagtgacc ttggcggggg cattggagga agtccggcag tgggacaatc cttcatccca
     1201 tcatcggtcc ctgcaacctt tgctccttca cctacacctg ctgtggtcag cagtggactg
     1261 aatgacctgt ttgaactctc cacagggata ggcatggcac ctggtggata tgtggctcct
     1321 aaggctgtct ggctacctgc agtaaaggct aaaggcttgg agatttccgg aacatttact
     1381 caccgccaag ggcacatcta tatggaaatg aacttcacca ataaagctct gcagcacatg
     1441 acagattttg caatccagtt taacaaaaat agctttggtg tcatccccag cactcctctg
     1501 gccatccata caccactgat gccaaaccag agcattgatg tctccctgcc tctcaatacc
     1561 ttgggcccag tcatgaagat ggaacctctg aataacctcc aggtggctgt gaaaaacaat
     1621 atcgatgtct tctacttcag ctgcctcatc ccactcaatg tgctttttgt agaagatggc
     1681 aaaatggagc gccaggtctt ccttgcaaca tggaaggata ttcccaatga aaatgaactt
     1741 cagtttcaga ttaaggaatg tcatttaaat gctgacactg tttccagcaa gttgcaaaac
     1801 aacaatgttt atactattgc caagaggaat gtggaagggc aggacatgct gtaccaatcc
     1861 ctgaagctca ctaatggcat ttggattttg gccgaactac gtatccagcc aggaaacccc
     1921 aattacacgc tgtcactgaa gtgtagagct cctgaagtct ctcaatacat ctatcaggtc
     1981 tacgacagca ttttgaaaaa cttaccggtc gccaccatgg tgagcaaggg cgaggagctg
     2041 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2101 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2161 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2221 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2281 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2341 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2401 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2461 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     2521 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2581 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     2641 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2701 gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgcgactc tagatcataa
     2761 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     2821 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     2881 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     2941 attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt
     3001 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3061 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3121 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3181 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3241 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3301 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3361 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3421 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3481 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3541 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     3601 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3661 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3721 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3781 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3841 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3901 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     3961 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4021 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4081 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4141 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4201 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4261 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4321 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4381 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4441 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     4501 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4561 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4621 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4681 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4741 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4801 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4861 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4921 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     4981 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     5041 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     5101 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     5161 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     5221 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5281 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5341 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5401 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5461 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5521 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5581 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5641 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5701 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5761 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5821 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5881 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5941 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6001 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6061 ccgccatgca tta
//
